This is a competitive U01 renewal application under the Integrative Neuroscience Initiative on Alcoholism- Neuroimmune (INIA-N) consortium (Notice# RFA-AA-16-004/5/6) to identify the role of immune signaling in ethanol dependence and develop new therapeutic strategies for the treatment of alcoholism. This U01 component will provide critical information on cellular mechanisms and neurobiological targets to other INIA- N investigators to bridge alcohol basic research with the human condition. Genetic evidence from INIA-N indicates that immune-related pathways, including interleukin-1? (IL-1?) and IL-10, are altered in alcohol dependence. Our recent results show that cytokines strongly influence neuronal function by modulating GABA neurotransmission in the central amygdala (CeA), a brain region involved in excessive drinking and ethanol dependence. The medial prefrontal cortex (mPFC) is also involved in the development of ethanol dependence and displays differences in its immune responses to chronic alcohol treatment; a stronger immune response is elicited in the cortex compared with the CeA. Our hypothesis is that alcohol exposure dysregulates neuroimmune mechanisms, leading to a general upregulation of proinflammatory elements and downregulation of antiinflammatory elements that contribute to altered synaptic transmission and alcohol-related behaviors. Thus, in this renewal, in addition to the focus on proinflammatory elements (such as IL-1?) we will investigate antiinflammatory systems (IL-10, IL-1ra) in the CeA and mPFC. In Aim 1 and 2, to induce ethanol dependence, we will use the chronic intermittent ethanol?two-bottle choice (CIE-2BC) paradigm in both male and female mice. We will apply behavioral, biochemical, and electrophysiological approaches to characterize dysregulation of the pro- and antiinflammatory mechanisms in alcohol-drinking animals. Aim 3 will provide behavioral and electrophysiological testing of the effects of ethanol-induced neuroimmune responses on physiological functions across species (rodents and nonhuman primates) and cellular and molecular mechanisms of the candidate drugs that are identified by other INIA-N projects in selected animal models. Thus, we anticipate that our studies will identify the role and mechanisms of action of neuroimmune factors in excessive alcohol drinking. Our collaborations with the other INIA-N PIs will promote replicability and translational aspects of our studies by testing our key targets in multiple species using multiple complementary approaches. The key personnel involved in this proposal possess all of the necessary expertise to accomplish this multidisciplinary program. Drs. M. Roberto and M. Bajo will be dedicated to the electrophysiology and biochemical studies, and Dr. A. Roberts will perform the behavioral studies. These key personnel each have extensive publication histories in their respective areas of expertise and ongoing collaborative interactions with many other INIA PIs that will be invaluable for the successful completion of the proposed experiments.